*Beads-oligo-dT19V: |--5'- /5Acryd/iSpPC/CGATGACG
T7 promoter: 5'-
W1 sequence: 5'-
RNA Ligation Oligo (RLO): 5'- /5Phos/
2nd RT primer: 5'-
PCR enrichment Primer 1: 5'-
PCR enrichment primer 2: 5'-
Illumina P5 adapter: 5'-
Illumina P7 adapter: 5'-
Read 1 sequencing primer: 5'-
Read 2 sequencing primer: 5'-
* barcode1 has variable length.
Acrydite-modified primer: 5'-/acrydite/iSpPC/ CGATGACG
BA19 oligo: 5'- BAAAAAAAAAAAAAAAAAAA -3'
FAM-PE1 probe: 5'-/6-FAM/ AGATCG
FAM-W1 probe: 5'-/6-FAM/
FAM-BA19 probe: 5'-/6-FAM/ BAAAAAAAAAAAAAAAAAAA -3'
Barcode 1 plates (384 of them in 384 individual wells): 5'-
Barcode 2 plates (384 of them in 384 individual wells): 5'- BAAAAAAAAAAAAAAAAAAA
|--5'-/acrydite/iSpPC/ CGATGACG TAATACGACTCACTATAGGG ATACCACCATGGCTCTTTCCCTACACGACGCTCTTC -3'
<-------- GAGAAAGGGATGTGCTGCGAGAAG GCTAGA[barcode1] CTCACTAACGAACACTGCGGAA -5' |--5'-/acrydite/iSpPC/ CGATGACGTAATACGACTCACTATAGGG ATACCACCATGGCTCTTTCCCTACACGACGCTCTTC -------->
3'- GCTACTGC ATTATGCTGAGTGATATCCC TATGGTGGTACCGAGAAAGGGATGTGCTGCGAGAAG GCTAGA[barcode1] CTCACTAACGAACACTGCGGAA -5' |--5'-/acrydite/iSpPC/ CGATGACGTAATACGACTCACTATAGGG ATACCACCATGGCTCTTTCCCTACACGACGCTCTTC CGATCT[barcode1] GAGTGATTGCTTGTGACGCCTT
|--5'-/acrydite/iSpPC/ CGATGACG TAATACGACTCACTATAGGG ATACCACCATGGCTCTTTCCCTACACGACGCTCTTC CGATCT[barcode1] GAGTGATTGCTTGTGACGCCTT
<-------- CTCACTAACGAACACTGCGGAA [8-bp barcode2] [6-bp UMI] A19B -5' |--5'-/acrydite/iSpPC/ CGATGACGTAATACGACTCACTATAGGG ATACCACCATGGCTCTTTCCCTACACGACGCTCTTC CGATCT[barcode1] GAGTGATTGCTTGTGACGCCTT ---------->
3'- GCTACTGC ATTATGCTGAGTGATATCCC TATGGTGGTACCGAGAAAGGGATGTGCTGCGAGAAG GCTAGA[barcode1] CTCACTAACGAACACTGCGGAA [8-bp barcode2] [6-bp UMI] A19B -5' |--5'-/acrydite/iSpPC/ CGATGACGTAATACGACTCACTATAGGG ATACCACCATGGCTCTTTCCCTACACGACGCTCTTC CGATCT[barcode1] GAGTGATTGCTTGTGACGCCTT [8-bp barcode2] [6-bp UMI] T19V -3'
|--5'-/acrydite/iSpPC/ CGATGACG TAATACGACTCACTATAGGG ATACCACCATGGCTCTTTCCCTACACGACGCTCTTC CGATCT[barcode1] GAGTGATTGCTTGTGACGCCTT [8-bp barcode2] [6-bp UMI] T19V -3'
5'- XXXXXXXXXXXXXXXXXXXB(A)n <-----V(T)19 [6-bp UMI] [8-bp barcode2] TTCCGCAGTGTTCGTTAGTGAG [barcode1] TCTAGCCTTCTCGCAGCACATCCCTTTCTC GGTACCACCATAGGGATATCACTCAGCATAAT GCAGTAGC -5'--|
5'- XXX...XXXB(pA) [6-bp UMI] [8-bp barcode2] AAGGCGTCACAAGCAATCACTC [barcode1] AGATCGGAAGAGCGTCGTGTAGGGAAAGAG CCATGGTGGTATCCCTATAGTGAGTCGTATTA CGTCATCG XXX...XXXV(dT)[6-bp UMI] [8-bp barcode2] TTCCGCAGTGTTCGTTAGTGAG [barcode1] TCTAGCCTTCTCGCAGCACATCCCTTTCTC GGTACCACCATAGGGATATCACTCAGCATAAT GCAGTAGC -5'--| ↵ IVT starts from here
5'- G AUACCACCAUGGCUCUUUCCCUACACGACGCUCUUC CGAUCU[barcode1] GAGUGAUUGCUUGUGACGCCUU [8-bp barcode2] [6-bp UMI] (dU)VXXX...XXX -3'
5'- G AUACCACCAUGGCUCUUUCCCUACACGACGCUCUUC CGAUCU[barcode1] GAGUGAUUGCUUGUGACGCCUU [8-bp barcode2] [6-bp UMI] (dU)VXXX...XXXAGATCGGAAGAGCGGTTCAGCAGGAATGCC -3'
5'- G AUACCACCAUGGCUCUUUCCCUACACGACGCUCUUC CGAUCU[barcode1] GAGUGAUUGCUUGUGACGCCUU [8-bp barcode2] [6-bp UMI] (dU)VXXX...XXXAGATCGGAAGAGCGGTTCAGCAGGAATGCC -3' <--------CAAGTCGTCCTTACGGCTCTG -5'
3'- C TATGGTGGTACCGAGAAAGGGATGTGCTGCGAGAAG GCTAGA[barcode1] CTCACTAACGAACACTGCGGAA [8-bp barcode2] [6-bp UMI] (pA)BXXX...XXXTCTAGCCTTCTCGCCAAGTCGTCCTTACGGCTCTG -5'
5'- AATGATACGGCGACCACCGAGATCTACAC TCTTTCCCTACACGA -----------> 3'-C TATGGTGGTACCGAGAAAGGGATGTGCTGCGAGAAG GCTAGA[barcode1] CTCACTAACGAACACTGCGGAA [8-bp barcode2] [6-bp UMI] (pA)BXXX...XXXTCTAGCCTTCTCGCCAAGTCGTCCTTACGGCTCTG -5' <-------------CAAGTCGTCCTTACGGCTCTGGC TAGAGCATACGGCAGAAGACGAAC -5'
5'- AATGATACGGCGACCACCGAGATCTACAC TCTTTCCCTACACGACGCTCTTC CGATCTNN....NN GAGTGATTGCTTGTGACGCCTT NNNNNNNN NNNNNN (dT)VXXX...XXXAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAGACCG ATCTCGTATGCCGTCTTCTGCTTG 3'-TTACTATGCCGCTGGTGGCTCTAGATGTG AGAAAGGGATGTGCTGCGAGAAG GCTAGANN....NN CTCACTAACGAACACTGCGGAA NNNNNNNN NNNNNN (pA)BXXX...XXXTCTAGCCTTCTCGCCAAGTCGTCCTTACGGCTCTGGC TAGAGCATACGGCAGAAGACGAAC -5'Illumina P5 PE1 barcode1 W1 8-bp 6bp cDNAPE2 Illumina P7 barcode2 UMI
5'- TCTTTCCCTACACGACGCTCTTC CGATCT------------------------------------------------> 3'-TTACTATGCCGCTGGTGGCTCTAGATGTG AGAAAGGGATGTGCTGCGAGAAG GCTAGANN....NN CTCACTAACGAACACTGCGGAA NNNNNNNN NNNNNN (pA)BXXX...XXXTCTAGCCTTCTCGCCAAGTCGTCCTTACGGCTCTGGC TAGAGCATACGGCAGAAGACGAAC -5'
5'- AATGATACGGCGACCACCGAGATCTACAC TCTTTCCCTACACGACGCTCTTC CGATCTNN....NN GAGTGATTGCTTGTGACGCCTT NNNNNNNN NNNNNN (dT)VXXX...XXXAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAGACCG ATCTCGTATGCCGTCTTCTGCTTG <-----------TCTAGCCTTCTCGCCAAGTCGTCCTTACGGCTCTGGC -5'
*Beads-oligo-dT19V: |--5'- /5Acryd/iSpPC/CGATGACG
T7 promoter: 5'-
PE1 adapter: 5'-
PE2 adapter: 5'-
PE2-N6 RT primer: 5'-
W1 sequence: 5'-
PE1 PCR primer: 5'-
PE2 PCR primer: 5'-
Illumina P5 adapter: 5'-
Illumina P7 adapter: 5'-
Read 1 sequencing primer: 5'-
Index sequencing primer: 5'- AGATCG
Read 2 sequencing primer: 5'-
* barcode1 has variable length.
Acrydite-modified primer: 5'-/acrydite/iSpPC/ CGATGACG
BA19 oligo: 5'- BAAAAAAAAAAAAAAAAAAA -3'
FAM-PE1 probe: 5'-/6-FAM/ AGATCG
FAM-W1 probe: 5'-/6-FAM/
FAM-BA19 probe: 5'-/6-FAM/ BAAAAAAAAAAAAAAAAAAA -3'
Barcode 1 plates (384 of them in 384 individual wells): 5'-
Barcode 2 plates (384 of them in 384 individual wells): 5'- BAAAAAAAAAAAAAAAAAAA
|--5'-/acrydite/iSpPC/ CGATGACG TAATACGACTCACTATAGGG ATACCACCATGGCTCTTTCCCTACACGACGCTCTTC -3'
<-------- GAGAAAGGGATGTGCTGCGAGAAG GCTAGA[barcode1] CTCACTAACGAACACTGCGGAA -5' |--5'-/acrydite/iSpPC/ CGATGACGTAATACGACTCACTATAGGG ATACCACCATGGCTCTTTCCCTACACGACGCTCTTC -------->
3'- GCTACTGC ATTATGCTGAGTGATATCCC TATGGTGGTACCGAGAAAGGGATGTGCTGCGAGAAG GCTAGA[barcode1] CTCACTAACGAACACTGCGGAA -5' |--5'-/acrydite/iSpPC/ CGATGACGTAATACGACTCACTATAGGG ATACCACCATGGCTCTTTCCCTACACGACGCTCTTC CGATCT[barcode1] GAGTGATTGCTTGTGACGCCTT
|--5'-/acrydite/iSpPC/ CGATGACG TAATACGACTCACTATAGGG ATACCACCATGGCTCTTTCCCTACACGACGCTCTTC CGATCT[barcode1] GAGTGATTGCTTGTGACGCCTT
<-------- CTCACTAACGAACACTGCGGAA [8-bp barcode2] [6-bp UMI] A19B -5' |--5'-/acrydite/iSpPC/ CGATGACGTAATACGACTCACTATAGGG ATACCACCATGGCTCTTTCCCTACACGACGCTCTTC CGATCT[barcode1] GAGTGATTGCTTGTGACGCCTT ---------->
3'- GCTACTGC ATTATGCTGAGTGATATCCC TATGGTGGTACCGAGAAAGGGATGTGCTGCGAGAAG GCTAGA[barcode1] CTCACTAACGAACACTGCGGAA [8-bp barcode2] [6-bp UMI] A19B -5' |--5'-/acrydite/iSpPC/ CGATGACGTAATACGACTCACTATAGGG ATACCACCATGGCTCTTTCCCTACACGACGCTCTTC CGATCT[barcode1] GAGTGATTGCTTGTGACGCCTT [8-bp barcode2] [6-bp UMI] T19V -3'
|--5'-/acrydite/iSpPC/ CGATGACG TAATACGACTCACTATAGGG ATACCACCATGGCTCTTTCCCTACACGACGCTCTTC CGATCT[barcode1] GAGTGATTGCTTGTGACGCCTT [8-bp barcode2] [6-bp UMI] T19V -3'
5'- XXXXXXXXXXXXXXXXXXXB(A)n <-----V(T)19 [6-bp UMI] [8-bp barcode2] TTCCGCAGTGTTCGTTAGTGAG [barcode1] TCTAGCCTTCTCGCAGCACATCCCTTTCTC GGTACCACCATAGGGATATCACTCAGCATAAT GCAGTAGC -5'--|
5'- XXX...XXXB(pA) [6-bp UMI] [8-bp barcode2] AAGGCGTCACAAGCAATCACTC [barcode1] AGATCGGAAGAGCGTCGTGTAGGGAAAGAG CCATGGTGGTATCCCTATAGTGAGTCGTATTA CGTCATCG XXX...XXXV(dT)[6-bp UMI] [8-bp barcode2] TTCCGCAGTGTTCGTTAGTGAG [barcode1] TCTAGCCTTCTCGCAGCACATCCCTTTCTC GGTACCACCATAGGGATATCACTCAGCATAAT GCAGTAGC -5'--| ↵ IVT starts from here
5'- G AUACCACCAUGGCUCUUUCCCUACACGACGCUCUUC CGAUCU[barcode1] GAGUGAUUGCUUGUGACGCCUU [8-bp barcode2] [6-bp UMI] (dU)VXXX...XXX -3'
5'- G AUACCACCAUGGCUCUUUCCCUACACGACGCUCUUC CGAUCU[barcode1] GAGUGAUUGCUUGUGACGCCUU [8-bp barcode2] [6-bp UMI] (dU)VXXX...XXX -3' <-------NNNNNNTCTAGCCTTCTCGCCAAGTCGTCCTTACGGCT -5'
5'- TCGGCATTCCTGCTGAACCGCTCTTCCGATCT XXX...XXXB(pA)[6-bp UMI] [8-bp barcode2] AAGGCGTCACAAGCAATCACTC [barcode1] AGATCGGAAGAGCGTCGTGTAGGGAAAGAG CCATGGTGGTATC -3'
5'- AATGATACGGCGACCACCGAGATCTACAC GGTCTCGGCATTCCTGCTGAAC ----------> 5'-TCGGCATTCCTGCTGAACCGCTCTTCCGATCT XXX...XXXB(pA)[6-bp UMI] [8-bp barcode2] AAGGCGTCACAAGCAATCACTC [barcode1] AGATCGGAAGAGCGTCGTGTAGGGAAAGAG CCATGGTGGTATC -3' <----------AGCACATCCCTTTCTC [6-bp sample index]TAGAGCATACGGCAGAAGACGAAC -5'
5'- AATGATACGGCGACCACCGAGATCTACAC GGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCT XXX...XXXB(pA)NNNNNN NNNNNNNN AAGGCGTCACAAGCAATCACTC NN....NN AGATCGGAAGAGCGTCGTGTAGGGAAAGAG NNNNNNATCTCGTATGCCGTCTTCTGCTTG -3' 3'-TTACTATGCCGCTGGTGGCTCTAGATGTG CCAGAGCCGTAAGGACGACTTGGCGAGAAGGCTAGA XXX...XXXV(dT)NNNNNN NNNNNNNN TTCCGCAGTGTTCGTTAGTGAG NN....NN TCTAGCCTTCTCGCAGCACATCCCTTTCTC NNNNNNTAGAGCATACGGCAGAAGACGAAC -5'Illumina P5 PE2 cDNA6bp 8bp W1 barcode1 PE1 6bpIllumina P7 UMI barcode2 sample index
5'- GGCATTCCTGCTGAACCGCTCTTCCGATCT ---------> 3'-TTACTATGCCGCTGGTGGCTCTAGATGTG CCAGAGCCGTAAGGACGACTTGGCGAGAAGGCTAGA XXX...XXXV(dT)NNNNNN NNNNNNNN TTCCGCAGTGTTCGTTAGTGAG NN....NN TCTAGCCTTCTCGCAGCACATCCCTTTCTC NNNNNNTAGAGCATACGGCAGAAGACGAAC -5'
5'- AGATCG GAAGAGCGTCGTGTAGGGAAAGAG -----> 3'-TTACTATGCCGCTGGTGGCTCTAGATGTG CCAGAGCCGTAAGGACGACTTGGCGAGAAGGCTAGA XXX...XXXV(dT)NNNNNN NNNNNNNN TTCCGCAGTGTTCGTTAGTGAG NN....NN TCTAGCCTTCTCGCAGCACATCCCTTTCTC NNNNNNTAGAGCATACGGCAGAAGACGAAC -5'
5'- AATGATACGGCGACCACCGAGATCTACAC GGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCT XXX...XXXB(pA)NNNNNN NNNNNNNN AAGGCGTCACAAGCAATCACTC NN....NN AGATCGGAAGAGCGTCGTGTAGGGAAAGAG NNNNNNATCTCGTATGCCGTCTTCTGCTTG -3' <-------------------------------------------TCTAGCCTTCTCGCAGCACATCCCTTTCTC -5'
5'- GGCATTCCTGCTGAACCGCTCTTCCGATCT ---------> 3'-TTACTATGCCGCTGGTGGCTCTAGATGTG CCAGAGCCGTAAGGACGACTTGGCGAGAAGGCTAGA XXX...XXXV(dT)NNNNNN NNNNNNNN TTCCGCAGTGTTCGTTAGTGAG NN....NN TCTAGCCTTCTCGCAGCACATCCCTTTCTC NNNNNNTAGAGCATACGGCAGAAGACGAAC -5'
5'- AAGGCGTCACAAGCAATCACTC -----> 3'-TTACTATGCCGCTGGTGGCTCTAGATGTG CCAGAGCCGTAAGGACGACTTGGCGAGAAGGCTAGA XXX...XXXV(dT)NNNNNN NNNNNNNN TTCCGCAGTGTTCGTTAGTGAG NN....NN TCTAGCCTTCTCGCAGCACATCCCTTTCTC NNNNNNTAGAGCATACGGCAGAAGACGAAC -5'
5'- AGATCG GAAGAGCGTCGTGTAGGGAAAGAG -----> 3'-TTACTATGCCGCTGGTGGCTCTAGATGTG CCAGAGCCGTAAGGACGACTTGGCGAGAAGGCTAGA XXX...XXXV(dT)NNNNNN NNNNNNNN TTCCGCAGTGTTCGTTAGTGAG NN....NN TCTAGCCTTCTCGCAGCACATCCCTTTCTC NNNNNNTAGAGCATACGGCAGAAGACGAAC -5'
5'- AATGATACGGCGACCACCGAGATCTACAC GGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCT XXX...XXXB(pA)NNNNNN NNNNNNNN AAGGCGTCACAAGCAATCACTC NN....NN AGATCGGAAGAGCGTCGTGTAGGGAAAGAG NNNNNNATCTCGTATGCCGTCTTCTGCTTG -3' <--------------------TTCCGCAGTGTTCGTTAGTGAG -5'